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Trakia Journal of Sciences, Vol. 4, No. 1, pp 33-36, 2006
ISSN 1312-1723 Original Contribution DETERMINATION OF TETRACYCLINE RESIDUES IN GREEK HONEY M. Saridaki-Papakonstadinou 1, S. Andredakis 1, A. Burriel *2, I. Tsachev 3
1Veterinary Laboratory Service, Ministry of Agriculture, Chania 73100, Crete, Greece
2Laboratory of Microbiology and Parasitology, Faculty of Veterinary Medicine, University of
3 Department of Microbiology, Infectious and Parasitic Diseases, Veterinary Faculty, Trakia
ABSTRACT
Liquid chromatography was used to detect tetracycline-derived residues in Greek honey. Of the examined samples 29% had these drug residues. 20.3% of these contained more than one of the observed tetracycline derivatives. Majority of samples contained residues to a proportion of 0.018-0.055 mg/kg of honey while some others had residues in excess of 0.100mg/kg. The findings show that tetracycline drug residues in Greek honey are present and possibly influence the naturally occurring protective properties of this medicinal food. Key Words: honey, tetracycline, liquid chromatography INTRODUCTION
protective properties in honey and may consequently affect adversely its antimicrobial
Honey is historically an essential ingredient of
activity. Specifically, these residues may
Mediterranean diet (Skidas and Lascaratos,
contribute to microbial resistance thereby
2001) and is also promoted currently around
altering the beneficial effects of the honey.
the world as a naturally protective product
The antimicrobial properties of honey appear
against various infections (Basson et al. 1994;
Drouin, 1999; Taormina et al. 2001) or burns
“inhibines” (Taormina et al. 2001; Zaghloul et
and wounds (Molan, 2002)1 The antimicrobial
al. 2001) that have intrinsic antibacterial
effects of honey, attributed to the presence of
actions, including activities against bacterial
antioxidant power (Taormina et al. 2001),
appear to be influenced by the type of honey.
2000; Ceyhan and Ugur, 2001; Dixon, 2003).
The dark coloured varieties of honey are
This honey protective property is making it a
thought to possess more inhibitory properties
preferred natural product for studying less
on selected pathogens than the light coloured
intrusive alternatives for protection of
ones. Other properties of honey, among which
is its anti-inflammatory activity, contribute to
rapid pain, oedema and exudate reductions.
of honey have also increased interest in the
maceration and adherence of dressings to the
determining honey quality, purity is the most
important. Various chemical contaminants or
residues of chemicals, including drugs used
strength of these properties, including the
researched vigorously and detection methods
are continuously improving (Vinas et al.
* Correspondence to: Burriel Angie DVM, MSc., MSc.,
naturally give rise to more classes of drug and
PhD., MRCVS; Mavromihali 154, Athens 11472, Greece; Tel.: 0030-6972-314842; E-mail:
Trakia Journal of Sciences, Vol. 4, No.1, 2006
SARIDAKI-PAPAKONSTADINOU M., et al.
Verzegnassi et al., 2003; Wang, 2004), with
the concomitant increase in proportion of
honey samples reported as positive (Heering
et al., 1998; Verzegnassi et al., 2003; Vinas et
passed through glass wool (SUPELCO) filters
before it was further purified through a Baker
organisms, suffer the consequence of disease,
column 10C18(SUPELCO) suitable for
and beekeepers often use either preventive or
separating organic compounds. The column
therapeutic antimicrobials to protect bee
was washed with 10 ml distilled water and
health and levels of honey production. The
further cleaned by the passing of high speed
tetracyclines, among other agents, have been
used consistently for treating foulbrood
were removed by passing through the column
caused by Paenibacillus larvae (Evans, 2003).
of 5 ml of ethyl acetate having 10% of MeOH.
The collected solutions were passed again
detectable amounts, possibly having through a 10 COOH Baker column, which considerable consequences to the consumer.
purification was achieved with the through
objective of state food inspecting agencies
passing of 0.01M and pH of 3.0 Oxalic acid
charged with the responsibility of ensuring the
safety level of honey. Various methods used
Merck) and Acetonitrile (LiChrosolv, Merck)
in detecting residual antimicrobials in foods
in a ratio of 5:4:1 respectively. The final
(Mitchell et al. 1998) may not be suitable for
honey, a product having naturally occurring
chromatographer using as positive controls
bacteriostatic substances (Ceyhan and Ugur,
samples having 0.30, 0.20, 0.05 and 0.100
2001; Zaghloul et al. 2001), but technological
improvements in available methods are giving
promising results (Vinas et al. 2004). Greek
honey, in order to be exported, must conform
Twenty nine percent of the examined samples
had drug residues of at least one of the
substances. To meet this requirement, the
tetracyclines. Of them 17 (20.3%) samples
Veterinary Laboratory Services of the Greek
had residues of more than one derivative. The
Ministry of Agriculture in Chania, Crete,
usually tests honey for tetracycline residues
before exportation. This effort, which started
samples was 0.018-0.057, 0.023-0.335, 0.018-
officially in 2003, is revealing interesting
0.190 and 0.013-0.393 for TC, OTC, DC and
results. The preliminary results reported here
CTC respectively. With an intra-laboratory
were produced using the slightly modified
reproducibility of 97%, 94%, 90% and 96%
method of Oka et al. (1987) for detecting
for TC, OTC, CTC DC, the positive samples
minute amounts of tetracycline derivatives.
detected and the areas they have originated
from are detailed in Table 1. The highest MATERIALS AND METHODS
proportion of positive samples had originated
Two hundred and fifty one samples of honey
from Thrace (53%), with the islands second
(37.5%) and Macedonia third (30.9%). Most
certification for exportation were mailed
of the positive samples were positive for OTC
between 2003 and 2004 to this laboratory.
(34 samples) with DC second (27 samples).
Producers wanting state permission to export
their product submitted these samples for
0.393 mg/kg of OTC and CTC respectively.
evaluation following government controls on
Honey from the islands appears to have the
the use of antibiotics for treating bee diseases.
lowest concentration of drugs and that of
Most samples were either pine tree or spring
classes used OTC residue was found in 14%
oxytetracycline (OTC), chlortetracycline
(CTC) and doxycycline (DC) were detected
according to Oka et al (1987) by a Hewlett
DISCUSSION
Packard 1100 liquid chromatographer (Hewlett Packard Inc) with a Diode Array
Very little to no information is available in the
column (25 cm x 4.6 mm, 5µm) at a light
tetracycline residues in honey, and this is the
Trakia Journal of Sciences, Vol. 4, No.1, 2006
SARIDAKI-PAPAKONSTADINOU M., et al.
internationally set acceptable amounts of
evidence of a rising problem with possible
tetracycline residues or standard methods for
sensitivity of the method used in the detection
Thus, one understands that, in the absence of
Table 1: Number of positive samples to each antimicrobial agent, area of origin and range of drug residues Pos (+) Neg (-) Antimicrobial (+) Area No SampleSamples s TC OTC DC CTC Total 251 73 178 12 36 25 21
The present findings compare well with those
suitable drugs or dose of various drugs used in
of others (Vinas et al. 2004) using a similar
combating bee infections or parasitic diseases.
methodology. The method used here detects
minute amounts of drug; thus it has high
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